Production of human antibodies using NOD/SCID/γcnull mice

ABSTRACT

The present invention provides an immunodeficient mouse (NOG mouse) suitable for engraftment, differentiation and proliferation of heterologous cells, and a method of producing such a mouse. This mouse is obtained by backcrossing a C.B-17-scid mouse with an NOD/Shi mouse, and further backcrossing an interleukin 2-receptor γ-chain gene-knockout mouse with the thus backcrossed mouse. It is usable for producing a human antibody and establishing a stem cell assay system, a tumor model and a virus-infection model.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 11/517,698 filed Sep. 8, 2006, nowabandoned, which is a divisional of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 10/221,549 filed on Sep. 10, 2002,now U.S. Pat. No. 7,145,055 issued Dec. 5, 2006, which is a §371 filingof PCT Application PCT/JP01/09401, filed Oct. 25, 2001, the entiredisclosures of which are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

A specification copy/computer-readable form of a sequence listing issubmitted electronically via EFS-Web and contains the filed namedCIE-0007D1C1_US_seqlist.txt, which is 876 bytes in size (measured inWindows XP), which was created on Sep. 27, 2010, and which is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method of producing an excellentmouse for engraftment of heterologous cells, a mouse produced by thismethod and use of the mouse.

BACKGROUND ART

Laboratory animals to which heterologous cells including human cells areengrafted are very important for analysis of onset mechanisms of variousdiseases and drug developments for the treatments or preventionsthereof, and development of animals as receptors therefor is one ofmajor themes in laboratory animal sciences. In particular, in recentyears, treatments etc. (known as regenerative medicine) in which tissuesor cells differentiated from stem cells are transplanted have receivedworld-wide attention, and therefore these animals are of increasingimportance.

The inventors of the present invention have continued to develop andimprove these laboratory animals. In particular, they made improvementsor the like on a nude mouse or a SCID mouse, and they have already fileda patent application (Japanese Patent Application Laying-Open (kokai)No. 9-94040) concerning an immunodeficient mouse etc. produced for thispurpose. Above all, an NOD/Shi-scid mouse and an NOD/LtSz-scid mousewhich exhibit multifunctional immunodeficiency (functional deficiency ofT cells and B cells, decline of macrophage function, reduction ofcomplement activity, reduction of natural killer (NK) activity etc.) arethe most noteworthy as laboratory animals suitable for engraftment ofheterologous cells. Since it became clear that they could be used forvarious types of research including stem cell differentiation andproliferation, the range of applications in which they are used hasincreased to the present level.

However, human cells are engrafted to the NOD/Shi-scid mouse at a highratio, but it is recognized that the engraftment capacity issubstantially varied.

In order to enhance the engraftment capacity of the NOD/Shi-scid mouse,it has already been revealed that reduction of NK activity in the mouseby administering anti IL-2Rβ chain antibodies (TMβ1), anti-asialo-GM1antibodies or the like is important. (Koyanagi, Y. et al., 1997.“Primary human immunodeficiency virus type 1 viremia and central nervoussystem invasion in a novel hu-PBL-immunodeficient mouse strain.” J Virol71:2417; Koyanagi, Y. et al., 1997. “High levels of viremia inhu-PBL-NOD-scid mice with HIV-1 infection.” Leukemia 11 Suppl. 3:109;Yoshino H, et al., 2000. “Natural killer cell depletion by anti-asialoGM1 antiserum treatment enhances human hematopoietic stem cellengraftment in NOD/Shi-scid mice.” Bone Marrow Transplant 26:1211-6.However, these antibodies are very expensive, and it is recognized thattheir efficacies vary between individuals. Further, when anti-asialo GM1antibodies are used, the administration thereof should be conducted withthe frequency of every eleventh day during the experiment period, andthus a degree of complexity is attached.

Therefore, Dr. Shultz, L. D. et al. of The Jackson Laboratory in theUnited States produced an NOD/LtSz-scid, β2m null (β2m (null)NOD/SCID)mouse (Kollet O, Peled A, Byk T et al., beta2 microglobulin-deficient(β2m(null))NOD/SCID mice are excellent recipients for studying humanstem cell function. Blood 2000; 95(10):3102-5) by crossing anNOD/LtSz-scid mouse having high engraftment capacity of human cells witha β2m KO mouse from which NK activity has been depleted.

With respect to the NOD/LtSz-scid, β2m null mouse, T cells, B cells andnatural killer (NK) cells are depleted, and the function of macrophagesand complements is reduced. However, other cells (e.g. dendritic cells)and factors (e.g. IFNγ) are also involved in the rejection oftransplanted heterologous cells or tissues.

Accordingly, a mouse which, compared to the NOD/LtSz-scid, β2m nullmouse, has no variation in heterologous cell engraftment capacity,requires no antibodies, and has excellent heterologous cell engraftmentis desirable.

DISCLOSURE OF THE INVENTION

Thus, the object of the present invention is to solve the above problemsand to provide a method of producing a mouse having excellentheterologous cell engraftment capacity and a mouse produced by the samemethod.

The present inventors have made intensive studies to solve the aboveproblems. As a result, they have obtained the findings that a mousewhich has no variation in engraftment capacity of heterologous cells andrequires no antibodies (that is, is suitable for the engraftment ofheterologous cells) can be obtained by backcrossing an NOD/Shi mousewith a C.B-17-scid mouse, and further backcrossing the thus obtainedmouse with an interleukin 2-receptor γ-chain (IL-2Rγ) gene-knockoutmouse. The present invention has been accomplished based on the abovefindings.

Namely, the present invention is as follows.

(1) A method of producing a mouse suitable for engraftment ofheterologous cells, comprising backcrossing a mouse B with a mouse A, asdescribed below:

A: a mouse obtained by backcrossing a C.B-17-scid mouse with an NOD/Shimouse; and

B: an interleukin 2-receptor γ-chain (IL-2Rγ) gene knockout mouse.

(2) The method of producing a mouse as described in (1), wherein themouse A is an NOD/Shi-scid mouse.

(3) The method of producing a mouse as described in (1) or (2), whereinthe mouse B is an IL-2RγKO mouse.

(4) A mouse produced by the method of producing a mouse described in anyof (1) to (3).

(5) A NOG (NOD/Shi-scid, IL-2RγKO) mouse having excellent engraftmentcapacity of heterologous cells, wherein both of functional T-cells andfunctional B-cells are deleted, macrophage function is reduced, NK cellsor NK activity are eliminated, dendritic cell function is reduced.

(6) The NOG mouse described in (4) or (5), wherein transplanted humanstem cells efficiently differentiate and proliferate without beingeliminated.

(7) A stem cell assay method comprising transplanting human stem cellsto the mouse described in any of (4) to (6) and analyzing thedifferentiated and proliferated cells.

(8) The stem cell assay method described in (7), comprising analyzingthe differentiation and proliferation of T-cells and B-cells.

(9) A method of proliferating human stem cells comprising:

-   -   transplanting and proliferating the human stem cells to the        mouse described in any of (4) to (6);    -   collecting the human stem cells from bone marrow of the mouse;        and    -   repeatedly transplanting the collected cells to the mouse        described in any of (4) to (6).

(10) The method of proliferating human stem cells described in (9),wherein the frequency of repeating is at least three times.

(11) Human stem cells obtained by the method of (9) or (10), wherein theobtained human stem cells have a purity of 99.7% or more.

(12) The method described in (9) or (10), wherein the human stem cellshave foreign genes introduced thereinto.

(13) The mouse described in any of (4) to (6), wherein the mouse iscapable of stably retaining human T-cells and B cells and producing ahuman antibody.

(14) A method of producing a human antibody comprising immunizing withan antigen the mouse described in any of (4) to (6) which retains humanT-cells and B-cells.

(15) A method of producing an antibody-producing cell line whichproduces a human antibody, comprising:

-   -   immunizing with an antigen the mouse described in any of (4)        to (6) which retains human T-cells and B-cells;    -   collecting from the mouse cells which produce the antibody        against the antigen; and establishing a cell line.

(16) A human tumor model mouse wherein the mouse is a mouse described inany of (4) to (6) and retains human tumor cells.

(17) The human tumor model mouse described in (16), wherein the humantumor cells are derived from HTLV-1 leukemia.

(18) The human tumor model mouse described in (16) or (17), wherein themouse has the human tumor cells at an auricle thereof.

(19) A method of screening an anticancer agent using the mouse describedin any of (16) to (18).

(20) A method of producing a human tumor model mouse comprisingtransplanting human tumor cells to the mouse described in any of (4) to(6).

(21) The method described in (16), wherein the human tumor cells arederived from HTLV-1 leukemia.

(22) The method described in (20) or (21), wherein the human tumor cellsare transplanted at an auricle of the mouse.

(23) A virus-infected model mouse wherein the mouse is a mouse describedin any of (4) to (6) and retains T cells infected with a T-tropic(T-cell affinity) virus as well as a macrophage-tropic virus.

(24) The virus-infected model mouse described in (23), wherein the virusis HIV.

(25) The virus-infected model mouse described in (23), wherein the virusis HTLV-1.

(26) A method of screening an antiviral agent wherein the method iscarried out using the mouse described in (23) to (25).

(27) A method of producing an immunodeficient mouse which hasengraftment capacity of enhanced heterologous cells compared with a NOGmouse, wherein the method is carried out using the mouse described in(3) to (6).

(28) The mouse described in (3) to (6), wherein the mouse is used forproducing an immunodeficient mouse which has enhanced engraftmentcapacity of heterologous cells compared with a NOG mouse.

Hereinafter, general embodiments of the present invention will bedescribed.

1. Production of a Mouse of the Present Invention

According to the present invention, a method of producing a mousesuitable for the engraftment of heterologous cells is characterized inbackcrossing a mouse B with a mouse A, as described below.

A: a mouse obtained by backcrossing a C.B-17-scid mouse with an NOD/Shimouse; and

B: an interleukin 2-receptor γ-chain gene knockout mouse.

Here, examples of the heterologous cells include cells or tissuesderived from mammals such as humans, mice, rats etc., particularly humanstem cells, lymphocytes or tumor cells etc. of humans, but not limitedthereto.

With respect to the mouse A, backcrossing the C.B-17-scid mouse with theNOD/Shi mouse is done in accordance with methods well-known to a personskilled in the art, for example, backcrossing by Cross Intercross method(Inbred Strains in Biomedical Research, M. F. W. Festing, 1979, ISBN0-333-23809-5, The Macmillan Press, London and Basingstoke). TheC.B-17-scid mouse is crossed with the NOD/Shi mouse, and the obtained F1mice are further crossed with each other. Then, the immunoglobulinamount in blood serum of the thus obtained F2 mice is measured forselecting a mouse, from which immunoglobulin cannot be detected. Theselected mouse is again crossed with a NOD/Shi mouse. Repeating thisprocess (Cross Intercross method) 9 times or more enables theaccomplishment of the backcrossing.

A NOD/Shi mouse and a C.B-17-scid mouse are both commercially availablefrom CLEA JAPAN, INC. Further, examples of mice obtained by crossingthese mice with each other include a NOD/Shi-scid mouse (also called asa NOD-scid mouse) (Japanese Patent Application Laying-Open (kokai) No.9-94040) which the present inventors have already established. Thismouse is purchased from CLEA JAPAN, INC., and can be used directly asthe mouse A. In addition, the present inventors possess, other than theones mentioned above, NOD/Shi mice and NOD/Shi-scid mice, which can besplit up and provided whenever the need arises.

Moreover, with respect to the mouse B, knockout of an interleukin2-receptor γ-chain (IL-2Rγ) gene is carried out in accordance withmethods well known to a person skilled in the art, for example, ahomologous recombination method using mouse ES cells (Capecchi, M. R.,Altering the genome by homologous recombination, Science, (1989) 244,1288-1292). After substituting a specific mouse-derived gene by ahomologous gene including a gene resistant to a drug, for exampleneomycin etc. at ES cell stage, the ES cells are inserted into afertilized egg, thereby accomplishing the gene-knockout.

Specifically, for example, gene clones containing a mouse IL-2Rγ areisolated, from a genome library of 129/SV mouse, using a humanIL-2RγcDNA as a probe. Using a fragment of 8.6 kb containing the fulllength of IL-2Rγ among the clones, a targeting vector is prepared. Thatis, PMCl-neo poly A which expresses a neomycin resistant gene, isinserted between exons 7 and 8 of IL-2R in the fragment, and also adiphtheria toxin-A gene is placed at 3′ side 1 kb away from exon 8.Next, the vector is made linear, and introduced into 1×10⁷ of E14 EScells by electroporation. Thereafter, ES clones which bring abouthomologous recombination in the culture solution including G418, areselected (confirmed by PCR or Southern method), and after injecting theES clones into blastocysts of C57BL/6 mice, they are transplanted intothe uteruses of foster parent mice. Chimeric mice born from the fosterparent mice are further crossed with C57BL/6 mice, thereby obtainingIL-2RγKO hetero mice wherein knockout is transduced to germ cells.

Alternatively, pre-established interleukin-2 receptor γ chain gene(IL-2Rγ) knockout mouse strain may directly be obtained for use fromsuppliers, and examples of the mouse strains include interleukin-2receptor γ chain (IL-2Rγ) knockout mice (Ohbo K, Suda T, Hashiyama M etal., Modulation of hematopoiesis in mice with a truncated mutant of theinterleukin-2 receptor gamma chain. Blood 1996; 87(3):956-67)) which wasproduced from IL-2RγKO mouse strains [Prof Kazuo Sugamura, Department ofMicrobiology and Immunology, Tohoku University School of Medicine].Incidentally, IL-2RγKO mice are presently stored in the embryopreservation bank of the applicants (Central Institute for ExperimentalAnimals) at the request of Prof. Sugamura, a producer of the mousestrain, and whenever the need arises they can be provided as frozenembryos or as thaw-reconstruction mice.

Further, backcrossing the mouse B with the mouse A can be carried out,in similar fashion as described above, according to conventional methodswell-known to a person skilled in the art. For example, in accordancewith the above backcrossing, that is, a NOD/Shi-scid mouse is crossedwith an IL-2RγKO mouse, and the obtained F1 mouse is backcrossed with anNOD/Shi-scid mouse, thereby accomplishing the backcross.

Furthermore, a mouse of the present invention is characterized in thatthe mouse is produced by the above method of the present invention. Themouse of the present invention is referred to as a NOG mouse (NOG mouse;NOD/Shi-scid, γc null mouse; NOD/Shi-scid, IL-2Rγ chain−/−mouse;NOD/Shi-scid, IL-2R(γc)^(null) mouse etc.)

The mouse of the present invention is a severe immunodeficient mouse inwhich has both of functional T-cells and functional B-cells are deleted,macrophage function are reduced, and NK cells or NK activity areeliminated. Therefore, when heterologous cells (e.g. human peripheralblood mononuclear leukocytes) are introduced into the mouse of thepresent invention, much higher ratios of engraftment and proliferationare observed even in comparison with conventional immunodeficient micewhich are subjected to anti NK antibody treatment. (See Example 1described below.) Further, dendritic cells of the mouse of the presentinvention are also functionally incompetent, and the production ofcytokine is remarkably reduced. Thus, the mouse of the present inventionhas the most excellent engraftment capacity of heterologous cells ascompared with conventional immunodeficient mice, and it is consideredeffective for analyses of various introduced heterologous cells(including stem cells, differentiated cells and cancer cells) which areengrafted in this mouse. Additionally, it is possible to use the mousein order to establish a pathologic model mouse and produce a humanantibody for HIV, HTLV-1 or cancer.

Hereinafter, the applications of the mouse of the present invention willbe described. Usually, the mouse to be used is preferably 8 to 12 weeksold, but not limited thereto.

2. Establishment of Human Stem Tell Assay System Using the Mouse of thePresent Invention

Using the mouse of the present invention, it is possible to establish ahuman stem cell assay system for examining factors and mechanisms whichare engaged in differentiation and proliferation of human stem cells.Also, it is possible to research various therapeutic products using thehuman stem cell assay system.

Introducing human stem cells into the mouse of the present inventionenables the establishment of the human stem cell assay system. Here,stem cells include, in addition to hematopoietic stem cells, stem cellsnot derived from the hematopoietic system, such as neural stem cellsetc. Human stem cells are identified by the existence of a cell surfacemarker which relates to a specific epitope site identified by anantibody, and for example, they can be isolated as CD34 positive cellsfrom e.g. human bone marrow, umbilical cord blood, peripheral blood etc.

Stem cells are suspended in a solution such as physiological saline,phosphate buffered physiological saline etc., which exerts no influenceon cells and living organisms, and 1×10⁴ to 1×10⁶ of cells areintravenously administered into the mouse, thereby carrying out thetransplantation.

Cells are collected, several weeks after transplanting, from each organsuch as peripheral blood, the spleen, bone marrow, the thymus etc. ofthe mouse to which the cells have been transplanted. The surfaceantigens of these cells are examined using e.g. FACS(Fluorescence-activated cell sorter), and thereby the differentiation ofthe transplanted cells is examined. In this case, examples of cellsurface antigen markers to be used as index include: CD34 which relatesto stem cells; CD3, CD4, CD8 etc. which relate to T-cells; CD10, CD19,CD20 etc. which relate to B-cells; CD5 etc. which relate to B1a cells;CD33 etc. which relate to myeloid cells; CD11c etc. which relate todendritic cells; CD45 etc. which relate to the whole leukocytes; CD11a,CD11b etc. which relate to macrophages; CD56 etc. which relate to NKcells; CD38 etc. which relate to plasma cells; CD41 etc. which relate toplatelets; and glycophorin A etc. which relate to erythrocytes.According to need, various related markers can be selected.

The production of cytokines such as interferon, interleukin, TNFα etc.in the collected cells, is measured by ELISA etc., and thereby thedifferentiation of the stem cells is examined.

Further, it is possible to conduct successive transplantations of humanstem cells using the mouse of the present invention. That is, trueself-replicable human stem cells can be obtained. Specifically, humanstem cells are transplanted in the mouse of the present invention, afterseveral weeks undifferentiated human stem cells are collected from bonemarrow of the mouse, and further the collected stem cells aretransplanted in the mouse of the present invention. By repeating thetransplantation and collection, human stem cells which are free fromother cells and have high purity can be obtained in large quantities. Bythe successive transplantations, human stem cells with at least 99% ormore purity, preferably 99.7% or more purity can be obtained.Conventional mice allow up to secondary transplantation, though themouse of the present invention enables more than two successivetransplantations.

For the treatment of leukemia or the like, human stem cells obtainedusing the mouse of the present invention can be transplanted to humans.Also, the mouse is usable for gene therapy targeting human stem cells byintroducing a foreign gene into human stem cells and transplanting themto the mouse of the present invention for proliferation. With the aid ofvirus vectors such as lentivirus vectors, retrovirus vectors, adenovirusvector, and adeno-associated virus vector, a gene can be introduced intostem cells. Examples of the genes to be used here include an ADA genefor adenosine deaminase deficiency (ADA) patients. After these genes areintroduced into human stem cells, the cells are transplanted to themouse of the present invention for proliferation and purification andthen administered to patients, thereby enabling gene therapy.

3. Production of Human Antibodies Using the Mouse of the PresentInvention

Using the mouse of the present invention, established human cell lineswhich produce human antibodies, and human antibodies can be obtained.The above-described human stem cells are transplanted to the mouse ofthe present invention, and cells responsible for immunity such asT-cells or B-cells are differentiated and proliferated. Alternatively,cells responsible for immunity such as human T-cells or B-cells aretransplanted to the mouse and engrafted in the mouse body, and therebyobtained is a mouse having the cells responsible for human immunity andcapable of producing human antibodies. When human stem cells aretransplanted, the differentiation and proliferation of T-cells andB-cells are realized in 6 to 8 weeks, enabling the production of humanantibodies.

By administering antigens to the mouse having human T-cells and B-cellsengrafted thereto and held, it is possible to obtain human antibodiesagainst the antigens and cells capable of producing the antibodies. Theadministration of the antigens to the mouse of the present invention maybe carried out by the same method as is conventionally used forimmunizing a mouse.

Human antibody producing cells can be collected from each organ of themouse, especially the spleen, lymph nodes etc. When the ratio of thehuman antibody producing cells is high, the collected cells can directlybe used for establishing a cell line. However, when the ratio is low, ifnecessary, purification may be carried out by e.g. the affinity columnmethod using anti-human B-cell antibodies. Further, it is desirable toeliminate mixed-in mouse cells by e.g. cytolysis method using anti mouseantibodies and complements.

The thus obtained human antibody producing cells are made into aestablished cell line by a transformation method using Epstein-Barrvirus (EBV), a cell fusion method wherein the cells are fused withsuitable proliferation viable cells, or the like. Then, obtainable is ahuman antibody-producing established cell line capable of multiplepassages while producing antibodies.

4. Production of a Pathologic Model Mouse with Tumor

In the mouse of the present invention, human tumors can be engrafted andproliferated, and an animal model of a human tumor can be obtained bytransplanting tumor cells to the mouse of the present invention. Forexample, the administration of the human tumor cells causes theproliferation thereof inside the mouse body, and thus a mouse having ahuman tumor can be obtained. Examples of the cells to be used in thiscase include subcultured lines of human tumor in a conventional nudemouse, and cell lines derived from HTLV-1 leukemia such as ED-40515(−),MT-1 and TL-Oml. In addition, human tumor tissues are chopped intopieces having a size of several mm, and these cancer tissue pieces maydirectly be transplanted and engrafted to the mouse of the presentinvention. In this case, the site of the mouse to which tumor cells ortissues are transplanted is not limited, but in the case of cells, theymay be transplanted intraperitoneally, intravenously or subcutaneouslyto the mouse and in the case of the tissues, they may be transplantedsubcutaneously to the mouse. Any subcutaneous site of the mouse may beacceptable such as subcutaneous gluteal region, but it is desirable totransplant them subcutaneously at an auricle or a dorsal region becausethe tumor can be checked without incision. Further, in order to obtainresults which reflect clinical effects of an anti cancer agent, it isdesirable to transplant them at the identical site as that for clinicaltest (in the case of colon cancer cells, the cells are to betransplanted to the colon). When the cells are transplanted, a tumor isformed within several weeks to several months. Specifically, when HTLV-1cells are transplanted subcutaneously at a posterior auricle, a tumor isformed in 2 weeks, thereby enabling expeditious production of apractical tumor model mouse.

Moreover, when a tumor is transplanted to the mouse of the presentinvention, metastases of tumor cells such as leukemic changes areobserved and the mouse is usable as a model animal for tumor metastasis.

Using the thus obtained human tumor model mouse, screening of an anticancer agent, antimetastatic drug etc. can be performed. As a methodtherefor, a candidate agent is administered to a mouse having a tumorformed, by a suitable method e.g. oral, transdermal administration orthe like. Then, observation on the size of the tumor, the size andnumber of metastatic focuses, the viability of the mouse etc., allowsjudgment on the effect of the drug.

5. Production of a Viral Infectious Disease Model Mouse

Use of the mouse of the present invention enables the obtainment of aviral infectious disease model mouse. Namely, by transplanting to themouse of the present invention cells which may be infected with a humanvirus, and infecting the cells with the virus, or transplanting cellsinfected with a virus, it is possible to obtain a viral infectiousdisease model animal, which has virus-infected cells engrafted and held.

In a conventional mouse, only an M-tropic virus which infectsmacrophages can proliferate, but the proliferation of T-tropic virusessuch as HIV, HTLV-1, which infects T-cells becomes possible using themouse of the present invention.

For example, 1×10⁷ to 1×10⁸ of human peripheral blood mononuclearleukocytes are intraperitoneally administered to the mouse of thepresent invention, and after several days some hundreds to thousands ofTCID₅₀ of HIV were inoculated, thereby obtaining a HIV-infected modelmouse having human cells infected with HIV. The HIV infection can bedetected through the expression of HIV antigens such as p24 positivecells as an index.

Instead of HIV, the inoculation of HTLV-1 enables the obtainment of anHTLV-1 infected model mouse.

Use of the animal model for disease obtained according to the presentinvention, allows in vivo research on proliferation mechanisms of HIV,HTLV-1 etc., further development of therapies for virus infections,screening of therapeutic products for virus infection, or the like.

6. Production of a Mouse Having Enhanced Engraftment Capacity ofHeterologous Cells Using a NOG Mouse

Use of the NOG mouse of the present invention enables the production ofa mouse having more enhanced heterologous cell engraftment. For example,such a mouse can be obtained by backcrossing a mouse wherein a generelating to the mouse immune system is knocked out with the NOG mouse ofthe present invention. Examples of the genes relating to the immunesystem include cytokine receptor gene, cytokine gene etc.

Further, by introduction of human cytokine gene, which relates to thedifferentiation and proliferation of human cells, or the like (e.g.hGM-CSF or hSCF etc.), it is possible to produce a mouse having moreenhanced heterologous cell engraftment. For instance, in accordance witha method of Pro. Natl. Acad. Sci. USA 77:7380-7384, 1980, or the like,the above gene is inserted into a pronuclear fertilized egg of themouse, and an individual having this introduced gene incorporatedthereinto is selected, thereby producing a mouse which expresses a humancytokine gene etc. Then, this mouse and the NOG mouse of the presentinvention were crossed with each other, thereby producing a mouse havingenhanced heterologous cell engraftment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an outline of backcrossing for producing a NOG mouse.

FIGS. 2A and 2B show time-course changes of human CD45 positive cellsand human CD41 positive cells, after the introduction thereof, inperipheral blood of the NOG mouse to which CD34 positive cells aretransplanted.

FIG. 3 shows the ratio of CD45 positive cells in the bone marrow andspleen of the NOG mouse to which CD34 positive cells are transplanted.

FIG. 4 shows the ratios of CD45 positive cells in peripheral blood ofmice in a comparative test between the NOG mouse and a β2 microglobulindeficient NOD-SCID mouse (NOD/LtSz-scid, β2m null mouse).

FIGS. 5A and 5B show FACS patterns of NK cells and dendritic cells inspleen cells obtained from each mouse strain.

FIGS. 6A, B and C show the results of ELISA for detecting the productionamount of cytokine under the stimulation of Listeria monocytogenesantigens in spleen cells obtained from each mouse strain.

FIG. 7 shows the removal of NK activity in the NOG mouse and theNOD/LtSz-scid, β2m null mouse.

FIG. 8 shows the results of FACS wherein bone marrow cells from primary,secondary and tertiary mice to which human CD34 positive cells have beentransplanted, are stained with human CD45.

FIGS. 9A and 9B show engraftment and differentiation of human cells inthe thymus of the NOG mouse to which human CD34 positive cells have beenintroduced.

FIGS. 10A and 10B show engraftment and differentiation of human cells inthe thymus of the NOG mouse to which human CD34 positive cells have beenintroduced.

FIGS. 11A and 11B show engraftment and differentiation of human cells inthe spleen of the NOG mouse to which human CD34 positive cells have beenintroduced.

FIGS. 12A and 12B show engraftment and differentiation of human cells inthe spleen of the NOG mouse to which human CD34 positive cells have beenintroduced.

FIGS. 13A and 13B show engraftment and differentiation of human cells inperipheral blood of the NOG mouse to which human CD34 positive cellshave been introduced.

FIGS. 14A and 14B show engraftment and differentiation of human cells inbone marrow of the NOG mouse to which human CD34 positive cells havebeen introduced.

FIGS. 15A, B and C show the ability of NOG mouse transplanted withumbilical cord blood (CB), bone marrow (BM) and peripheral blood stemcells (PBSC), respectively, to produce human antibodies.

FIG. 16 shows the tumor formations after transplanting LM-2-JCK to eachmouse strain.

BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described in detail by referring toExamples.

Example 1 Production of an Immunodeficient Mouse (NOG Mouse) with theDeletion of NK Activity and Declined Dendritic Cell Function,Examination of the Heterologous Cell Engraftment in the Mouse, andEstablishment of an Assay System of Human Stem Cells Using the Mouse

(1) Production of an Immunodeficient Mouse (NOG Mouse) with theElimination of NK Activity and Reduced Dendritic Cell Function

In order to obtain multifunctional immunodeficient mice with depleted NKactivity, interleukin-2 receptor γ chain knockout mice (IL-2RγKO mice)(8 week-old) which were transferred from Prof. Kazuo Sugamura(Department of Microbiology and Immunology, Tohoku University, School ofMedicine) were backcrossed with NOD-Shi-scid mice (8 week-old) whichwere kept in the Central Institute for Experimental Animals (alsoavailable from CLEA JAPAN, INC.), and thereby F1 mice having an IL-2Rγmutant gene introduced thereinto were produced. The introduction of themutant IL-2Rγ chain gene in the F1 mice was confirmed by PCRamplification and detection of the gene. Specifically, first, DNAs wereextracted by a DNA automatic extractor (MagExtractor manufactured byTOYOBO) from 100 μl of blood taken from ocular fundus of the F1 mice.PCR buffer solution containing 23.5 L of 1.5 mM MgCl₂, 0.4 mM dNTP andtwo sets of 25 μmol primers (the following primers PI and PIII were usedfor the determination of wild type, and a set of the following primersPI and PII were used for the determination of mutant type) was added to1.5 μL of this DNA, and PCR was conducted under the followingamplification conditions for the determination of whether IL-2Rγ chaingenes were wild type or mutant type.

(Primers)

PI: 5′-CTGCTCAGAATGATGCCTCCAATTCC-3′ (SEQ ID NO: 1) PII:5′-CCTGCGTGCAATCCATCTTGTTCAAT-3′ (SEQ ID NO: 2) PIII:5′-GATCCAGATTGCCAAGGTGAGTAG-3′ (SEQ ID NO: 3)

(PCR Amplification Conditions)

The conditions were heating at 94° C. for 5 minutes; 30 to 35 cycles of1 minute at 94° C., 1 minute at 55° C., and 1 minute at 72° C.; andthereafter heating at 72° C. for 10 minutes.

The PCR products obtained by the above PCR were subjected toelectrophoresis in 2% agarose gel, and measured according to the size ofthe coloring band detected after ethidium bromide stain. The sizes ofthe bands, about 660 bp for wild type and about 350 bp for mutant type,were observed.

(Backcrossing)

Next, the F1 Mice Having the Mutant IL-2Rγ Gene Introduced Thereintowere crossed with NOD/Shi-scid mice, thereby obtaining F2 mice. Further,by detecting the introduction of the mutant IL-2R γ chain gene into theF2 mice in the same manner as above, and detecting immunoglobulins inserum by an immunodiffusion method, mouse individuals which had themutant IL-2R γ chain gene and had a homozygous scid gene were selected.Thereafter, the mouse individuals were crossed with NOD/Shi-scid mice,and among the born mice, mice having mutant IL-2R γ chain gene werefurther crossed with NOD/Shi-scid mice.

The above backcross was repeated at least 9 times, thereby producing NOG(NOG) mice (FIG. 1 shows the outline). Here, since the IL-2R γ chaingene exists on an X chain chromosome, it is effective to use maleIL-2RγKO mice.

(2) Examination on Engraftment Capacity of Heterologous Cells in NOGMice

Next, using the NOG mice obtained by the above crossing and conventionalimmunodeficient mice, NOD/Shi-scid mice, examinations were made on thelevel of impact that anti-NK antibody treatment has on engraftment ofheterologous cells in these mice.

(Anti-IL-2 Receptor β Chain Monoclonal Antibody)

Anti-IL-2 receptor β chain monoclonal antibodies (clone TM β1) wereproduced from hybridomas produced and provided by Prof. MasayukiMiyasaka, School of Medicine, Osaka University (Tanaka T, Tsudo M,Karasuyama H et al., A novel monoclonal antibody against murineIL-2Receptor beta-chain. Characterization on of receptor expression innormal lymphoid cells and EL-4 cells. J Immunol 1991; 147(7):2222-8). Inparticular, the hybridomas were intraperitoneally administered toBALB/cA-nu mice and collected from ascites after several weeks.

One mg per mouse of the antibodies was intraperitoneally administered tofive NOD/Shi-scid mice (8 to 12 week-old) and three NOG mice (8 to 12week-old). Further, as controls with no administration of theantibodies, physiological saline was administered to four NOG mice (8 to12 week-old).

Moreover, human peripheral blood lymphocytes were collected by use ofdensity gradient centrifugation using Lymphoprep, from blood taken fromvolunteers.

1×10⁷ of the obtained human peripheral blood mononuclear leukocytes wereintraperitoneally administered to the above mice on the second day afterthe administration of TM β1.

The mice were sacrificed 2 weeks after the administration of humanperipheral blood lymphocytes, and the ascites containing the wholeperitoneal exudates cells were fully washed with RPMI-1640 culturemedium and thereby collected. Out of all the peritoneal cells, all theperitoneal exudates cells were counted by flow cytometry, and dependingon their amounts, the engraftment and proliferation of human cells intomice were determined. The same operations were conducted for controlmice with no administration of the antibodies. Their results are shownin Table 1.

TABLE 1 Impact of anti-NK antibodies (TMβ1) on engraftment andproliferation of human peripheral blood mononuclear leukocytes inNOD/Shi-scid mice and NOG mice Number of collected Human cells % TMβ1Number of cells (×10⁶, HLA+ Mouse strain treatment mice distribution)(distribution) hCD4⁺ hCD8⁺ NOD/Shi-scid + 5  4.8 (2.77-6.8) 41.1(4.2-65)   ND ND NOG + 3 11.1 (8.3-15.0) 61.9 (47.4-74.6) 19.8 28.2 − 411.2 (7.9-20)   63.3 (51.4-69.7) 34.3 25.9

It is clear from the results shown in Table 1 that extremely high ratiosof human cells were differentiated, engrafted and proliferated, evenwithout the treatment of the antibodies, in NOG mice to which humanperipheral blood mononuclear leukocytes were introduced, as comparedwith conventional TM β1 treated mice.

(Anti Asialo-GM1 Antibody)

Next, using NOG mice and NOD/Shi-scid mice, examinations were made onthe level of impact, which would be given by anti asialo-GM1 antibodies(AGM1) (rabbit) (Wako Pure Chemical Industries, Ltd., 014-09801), onengraftment capacity of cells derived from human umbilical cord blood inthese mice.

First, 2.4 Gy of X ray were irradiated on NOG mice (8 to 12 week-old)and NOD/Shi-scid mice (8 to 12 week-old) which were numbered asindicated in Table 2. Then, 20 μL per mouse of anti asialo-GM1antibodies diluted with 400 μL PBS were administered to these mice justbefore the administration of the cells derived from human umbilical cordblood. Also, physiological saline was intraperitoneally administered tocontrol mice (No. 5, 6, 10, 11, 15, 16 mice in Table 2) withoutadministration of the antibodies.

With respect to human umbilical cord blood CD34 positive cells,mononuclear leukocytes were collected by Ficoll-Hypaque gradientcentrifugation from umbilical cord blood taken from volunteers from whomapproval was each obtained in advance. Further, CD34 positive cells wereisolated by Dynabeads M-450 CD34 and DETACHaBEAD CD34 (Dynal As, Oslo,Norway). (Ueda T, Tsuji K, Yoshino H et al., Expansion of humanNOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand,thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105(7):1013-21)

1×10⁵ of the obtained CD34 positive cells derived from human umbilicalcord blood were introduced through tail veins of the mice immediatelyafter the administration of anti asialo-GM1 antibodies.

The mice were sacrificed 4 weeks after the administration of CD34positive cells derived from human umbilical cord blood, their peripheralblood was collected, and human cells (CD45⁺) in mononuclear leukocytesand human platelets (CD41⁺) in the whole blood were counted by flowcytometry. Depending on their amounts, the engraftment capacity andproliferation of human cells into mice were determined. The sameoperations were conducted for control mice without administration of theantibodies. Their results are shown in Table 2.

TABLE 2 Differentiation and proliferation of introduced human umbilicalcord blood CD34⁺ in NOG mice 4 weeks after introduction No. of No. ofNo. of No. of Mouse AGM1 introduced Days after Leukocytes erythrocytesplatelets hCD45⁺ hCD45⁺ hCD41⁺ hCD41⁺ No. Mouse Strain treatment CD34⁺cells transplant (10²/ul) (10⁴/ul) (10⁴/ul) (%) (/ul) (%) (/ul) 1NOD/Shi-scid + 100000 31(4 wk) 12 782 134 0.19 2 0.001 20 2NOD/Shi-scid + 100000 31(4 wk) 12 809 147 0.94 11 0.022 318 3NOD/Shi-scid − 100000 31(4 wk) 10 758 104 0.48 5 0.004 41 4 NOD/Shi-scid− 100000 31(4 wk) 14 773 109 3.24 45 0.166 1814 5 NOG − 100000 31(4 wk)12 713 89.3 5.84 70 0.255 2274 6 NOG − 100000 31(4 wk) 17 749 108 9.29158 0.324 3498 7 NOD/Shi-scid + 100000 29(4 wk) 10 734 139 1.82 18 0.056772 8 NOD/Shi-scid + 100000 29(4 wk) 10 787 118 1.51 15 0.094 1105 9NOD/Shi-scid − 100000 29(4 wk) 7 722 132 0.97 7 0.059 776 10 NOG −100000 29(4 wk) 36 602 218.1 1.84 66 0.170 3709 11 NOG − 100000 29(4 wk)10 725 111 9.28 93 0.518 5748 12 NOD/Shi-scid + 100000 28(4 wk) 7 69692.4 1.74 12 0.007 63 13 NOD/Shi-scid + 100000 28(4 wk) 10 787 96 0.40 40.008 73 14 NOD/Shi-scid − 100000 28(4 wk) 10 710 82.6 0.41 4 0.021 17315 NOG − 100000 28(4 wk) 2 684 103 8.52 17 0.101 1040 16 NOG − 10000028(4 wk) 2 773 86.5 14.98 30 0.196 1692 Average NOD/Shi-scid(AGM1+) 1.1010.52 0.03 391.74 NOD/Shi-scid(AGM1−) 1.27 15.25 0.06 701.14 NOG 7.1161.99 0.22 2565.96

It is clear from the results shown in Table 2 that extremely high ratiosof human cells were differentiated, engrafted and proliferated, evenwithout the treatment of the antibodies, in NOG mice to which CD34positive cells derived from human umbilical cord blood were introduced,as compared with conventional mice which were treated with antiasialo-GM1 antibody.

(3) Establishment of Assay System of Human Stem Cells Using NOG Mice

Mice to be used here were NOD/Shi-scid mice and NOG mice of the presentinvention. 1×10⁵ of CD34 positive cells taken from human umbilical cordblood (CB) were transplanted to the above mice which had received 2.4 Gyof radiation.

Human cells in peripheral blood, bone marrow, spleens, and thymuses wereanalyzed by FACS. The ratios of CD45 positive cells in peripheral blood8 weeks after the transplant were 37% for NOG mice and 7% forNOD/Shi-scid mice. In bone marrow, the ratios were 65% and 20%,respectively. In addition to a high ratio of chimerism, it was foundthat differentiations occurred into various cell lines including B and Tlymphocytes in peripheral blood, bone marrow, spleens, and thymuses ofthe NOG mice 3 month after the transplant. It was observed that humanCD33⁺ bone marrow cells, CD19⁺B cells, CD3⁺T cells, CD56⁺NK cells,CD41a⁺huge nucleoplasm, and glycophorin A⁺ erythrocytes were present inbone marrow. Further, it is interesting to note that though there wereobserved a small number of human T cells in the thymuses, CD4⁺/CD8⁻ Tcells, CD4⁻/CD8⁺T cells, and CD4³⁰/CD8⁺T cells were observed in thespleens. This indicates that human hematopoietic stem cells aredifferentiated and proliferated into mature T cells at sites apart fromthe thymus.

Umbilical cord blood was obtained at birth of a healthy newborn from anormal pregnant mother from whom approval was obtained in advance. Theumbilical cord blood was heparinized and preserved, and treated by theoperation described below within 24 hours after collecting. Then, it wasused for a transplant test.

The purification of CD34 positive cells was conducted as follows. Theheparinized umbilical cord blood was diluted two times with a buffersolution prepared by mixing phosphate buffered saline with 5% fetalbovine serum, and thereafter mononuclear leukocytes were isolated usingFicoll. From the isolated mononuclear leukocytes, CD34 positive cellswere purified by use of Dynabeads™ M-450 CD34 available from DynalBiotech, Ltd. Although the method therefor is as instructed by DynalBiotech, Ltd., it is summarized as follows. Using phosphate bufferedphysiological saline containing 2% bovine serum albumin and sodiumcitrate, the concentration of the mononuclear leukocytes was adjusted soas to be 4×10⁷/mL. 100 μL of well-suspended Dynabeads CD34 was added per1 mL of mononuclear leukocyte suspension, and they were blended andreacted with each other over ice for 30 minutes. Beads which formedrosettes with cells were collected using a magnet. Subsequently, to theBeads forming rosettes with cells, a required amount of Detachabead™CD34 was added and reacted therewith, blending at 37° C. for 15 minutes.As CD34 positive cells were liberated from the Beads, a magnet was usedto remove only the Beads, thereby obtaining CD34 positive cells.

For transplanting, 8 to 12 week-old NOD/Shi-scid mice, NOG mice, andNOD/LtSz-scid, β2m null mice, all raised in an SPF (specific pathogenfree) environment were used. These mice were raised in the animalexperiment facility of Kyoto University, School of Medicine under theregulations of the facility.

In a comparative test for the former 2 types of mice, the mice weretwice irradiated with 1.2 Gy gamma ray (gamma cell ¹³⁷Cs), and 100,000of CD34 positive cells per mouse were transplanted through the tailvein. For the NOD/Shi-scid mice, 200 μg of anti asialo GM1 antibodies(Wako Pure Chemical Industries, Ltd.) were intraperitoneallyadministered immediately before the transplant and every eleventh dayafter the transplant.

In a comparative test between NOG mice and NOD/LtSz-scid, β2m null mice,the mice were twice irradiated with 1.2 Gy gamma ray in the same way asabove, and thereafter 40,000 or 10,000 of CD34 positive cells weretransplanted through the tail vein.

After transplantation, 732 mg/L of neomycin sulfate were added to themice's drinking water for protection against infection.

The mice were ether-anaesthetized at moments as indicated, and thenperipheral blood was collected from the orbital venous plexus formeasuring the positive ratio of human hemocytes by use of flowcytometry. Although the flow cytometry was conducted in accordance witha widely-used general method, it is summarized as follows. The collectedblood was instantly blended well with EDTA-2Na and allowed to stand atroom temperature until analysis. An optimum amount of antibodies wasadded to and reacted with 50 to 100 μL of the whole blood at 4° C. for30 minutes. Thereafter, hemolysis and immobilization were effected usingFACS solution (Becton Dickinson and Co.), and the ratio was measuredusing FACS Calibur (Becton Dickinson and Co.). In the case of bonemarrow and spleen, the mice were sacrificed with cervical vertebradislocation and femora and spleens were eviscerated. Then, bone marrowand splenic cells were each liberated in culture solution containing 5%fetal bovine serum, and thereafter as cell suspensions, they weretreated in the same manner as peripheral blood and analyzed by flowcytometry.

Antibodies to be used for the analysis were FITC conjugated anti humanCD45 antibodies, PE conjugated anti human CD10 antibodies, PE conjugatedanti human CD33 antibodies, PE conjugated anti human CD3 antibodies, PEconjugated anti human CD34 antibodies, PE conjugated anti human CD41antibodies (Beckton, Dickinson and Co.), PC5 conjugated anti human CD38antibodies, PC5 conjugated anti human CD56 antibodies, PC5 conjugatedanti human CD19 antibodies (Immunotech), APC conjugated anti mouse CD45antibodies, and FITC conjugated anti mouse CD41 antibodies (BDPharmingen).

With respect to human CD45 positive cells and human CD41 positive cellsin mouse peripheral blood, changes in the ratios (%) and the absolutenumbers (/μL) were investigated every fourth week until the 12th week(FIG. 2A and FIG. 2B). As shown in FIGS. 2A and 2B, significantly highratios and absolute numbers were observed for NOG mice. The mice weresacrificed from 12 weeks onward after the transplant, and when theratios of human CD45 positive cells in bone marrow and spleens wereinvestigated in the same way as above, the NOG mice had very highpositive ratios. (FIG. 3)

In the comparative test between NOG mice and NOD/LtSz-scid, β2m nullmice, the positive ratios of human CD45 positive cells in mouseperipheral blood were investigated (FIG. 4). As shown in FIG. 4, the NOGmice had a higher positive ratio of human cells and exhibited a betterengraftment capacity of human cells than NOD/LtSz-scid, β2m null mice.

Example 2 Examinations on Functional Incompetence of Dendritic Cells ofNOG Mice in NK Activity and Cytokine Production

Three female NOG mice (10 to 12 week-old) of the present invention, fourfemale and four male NOD/Shi-scid mice (10 to 12 week-old) obtained bybackcrossing C.B-17-scid mice with NOD/Shi mice, and two female and twomale NOD/LtSz-scid, β2m null mice (10 to 12 week-old) (or referred to asnull β2m(null)NOD/LtSz-SCID, β2m microglobulin deficient NOD/SCID mice;produced by Dr. Shultz, L. D. et al. of The Jackson Laboratory Kollet O,Peled A, Byk T et al., beta2 microglobulin-deficient(β2m(null))NOD/SHI-SCID mice are excellent recipients for studying humanstem cell function. Blood 2000; 95(10):3102-5) were used for thefollowing examinations.

After the NOD/Shi-scid mice were treated with anti asialo GM1 antibodies(αAGM), spleen cells were collected. The obtained cells were dividedinto two groups, and CD11c antigen positive cells (regarded as dendriticcells) were removed from one of them using a magnetic cell sorter(MACS). Likewise, splenic cells were collected from non-treatedNOD/LtSz-scid, β2m null mice and NOG mice. A small amount of these cellswas taken from each of them and provided for FACS analysis. Further,these four kinds of cells were divided into three groups: group I: NOGmice (3 mice); group II: AGM non-treated NOD/Shi-scid mice (2 males),αAGM treated NOD/Shi-scid mice (3 mice), and NOD/Shi-scid mice (3 mice)which were treated with αAGM and further from which CD11c were removed;and group III: NOD/LtSz-scid, β2m null mice (4 mice). They were adjustedto have a cell concentration of 1×10⁷/ml in RPMI-1640, and 100 μl ofeach of them were dispensed into a well, stimulated by an equivalentamount of Listeria monocytogenes (LM) antigens, and cultured (usingTriplicate). Since LM is 10¹¹ Units/mL, it was diluted for use so as tobe 2×10⁷ Units/mL with RPMI-1640. In this case, specimens with no LMaddition were used as a control. After 24 hours, the supernatants werecollected, and the amounts of cytokine such as IFN-γ were determined byELISA.

Subclasses (in particular, CD11c, CD11b) in spleen cells were analyzedby FACS.

FITC-labeled CD3 and biotine-labeled Pan NK(DX5) were used as a T-cellmarker and an NK cell marker, respectively. FITC-labeled anti-CD11c andPE-labeled anti-CD11b were used as a macrophage and dendritic cellmarker.

Further, NK activity in NOG, C.B-17-scid, NOD/Shi-scid, andNOD/LtSz-scid, β2m null mice (10 to 12 week-old) was examined bycytotoxicity test targeting ⁵¹Cr-labeled NK susceptible YAC-1 cells.Namely, splenic cells were isolated from the mice and mixed and culturedwith ⁵¹Cr-labeled YAC-1 cells at various ratios therebetween. Afterculturing at 37° C. for 4 hours under 5% CO₂, the radioactivity insupernatants was measured by a liquid scintillation counter. The NKactivity is indicated according to the following calculation method.% specific cytotoxicity=(specific radioactivity−backgroundradioactivity)/(maximum radioactivity−background radioactivity)×100

FIGS. 5A and 5B show FACS patterns of the spleen cells obtained fromeach mouse strain.

NK cells were detected from NOD/Shi-scid and NOD/LtSz-scid, β2m nullmice, though NK cells were not detected at all from NOD/Shi-scid micetreated with anti asialo GM1 antibody and NOG mice. CD11c positivecells, which were used as a dendritic cell marker were detected from allthe mice at extremely high ratios. It was confirmed that CD11c positivecells were almost completely removed from the splenic cells of theNOD/Shi-scid mice treated with anti asialo GM1 antibody by magneticbeads.

FIGS. 6A, 6B and 6C show detection results by ELISA on the productionamount of cytokine in the above splenic cells under the LM stimulation.The following was found: although IFN γ production was observed inNOD/Shi-scid mice not treated with anti asialo GM1 antibody andNOD/LtSz-scid, β2m null mice, there was no detection from NOG mice; and,as with NOG mice, by removing CD11c positive cells from the NOD/Shi-scidmice treated with anti asialo GM1 antibody, IFN γ production was notobserved.

FIG. 7 shows NK activity of the splenic cells obtained from each mousestrain.

In comparison with C.B-17-scid mice, reduction of NK activity inNOD/Shi-scid mice was observed, but absolutely no NK activity wasobserved in NOG mice and NOD/LtSz-scid, β2m null mice.

In view of the foregoing, it became clear that NOG mice andNOD/LtSz-scid, β2m null mice completely lost their NK activity. However,according to the result of FACS analysis, it became clear that the NOGmice lost NK cells, though the NOD/LtSz-scid, β2m null mice had NK cellspresent therein but lost NK activity.

Further, it was revealed that the functional incompetence of thedendritic cells was a cause for the cytokine production decline insplenic cells, which was observed in the NOG mice. On the other hand, itwas indicated that the NOD/LtSz-scid, β2m null mice had almost the samepatterns of FACS and cytokine production as the anti asialo GM1antibody-treated NOD/Shi-scid mice. It was obvious that theNOD/LtSz-scid, β2m null mice lost NK cells and their dendritic cellswere normal.

Example 3 Successive Transplant of Human Stem Cells Using NOG Mice

In order to examine self-replicability of gene-introduced human stemcells, umbilical cord blood stem cells were transplanted to NOG mice,and it was examined whether or not secondary or tertiary transplant ispossible.

Umbilical cord blood was obtained from pregnant women, with theirconsent and approval, for use in tests. After mononuclear cells wereisolated from umbilical cord blood using Ficoll-Hypaque (Lymphoprep,1.077±0.001 g/ml; Nycomed, Oslo, Norway), CD34 positive cells werepurified using a CD34 positive separation column (MACS, Miltenyi Biotec,Glodbach, Germany). The CD34 positive cells had a purity of 96±3%.

Lentivirus vector pCS-CG was a vector which enables a GFP gene to beexpressed by a CMP promoter, and it was provided by Dr. Miyoshi(Immunology, Medical Branch, University of Tsukuba). After umbilicalcord blood CD34 positive cells were cultured for 24 hours in serum-freemedium StemPro TM-34SFM (Gibco BRL) containing 50 ng/mL of each TPO, SCFand Flk-2/Flt-3 ligand (FL), they were subjected to five-hour infectionof recombinant lentivirus CS-CG by MO130 (Kawada H et al., Exp. Hematol.27:904-915, 1999). Thereafter, cells were washed and subjected to 5-dayextracorporeal amplification culture in the same serum-free medium asabove in the presence of murine bone marrow stroma cells HESS-5 (Oki Met al., Exp. Hematol. In press. 2001).

After 7 week-old NOD/Shi-scid mice were subjected to 3 Gy radiation,3×10⁵ of gene-introduced cells which were amplified by culture, wereintroduced through the tail vein. After 6 weeks, surface antigens ofmurine bone marrow cells were analyzed, and 1×10⁷ of cells wereintroduced into irradiated NOG mice (secondary transplant). After 6weeks, the same analysis was conducted, and 1×10⁷ of cells wereintroduced into irradiated NOG mice (tertiary transplant). Further,after 6 weeks, the same analysis was conducted.

The analysis of surface antigens was conducted using FACS Calibur(Becton, Dickinson and Co.). The antibodies used here were FITC-labeledanti-human CD45 (T-200), PE-labeled anti human CD2 (39C1.5), CD3(UCHT1),CD4(SK3), CD14(LeuM3), CD19(4G7), CD20(2H7), CD33(WM53), CD41(P2),CD56(N901), and glycophorin A (KC16), and all of them were purchasedfrom Becton, Dickinson and Co.

The gene introduction efficiency into CD34 positive cells was 40±5%(n=5). FIG. 8 shows the results of FACS wherein mouse bone marrow cellsof primary, secondary, and tertiary transplant mice were stained withhuman CD45. It was observed that gene-introduced human cells existed inall the mice.

By using NOG mice of the present invention as a host, the inventorsbecame the first in the world to successfully accomplish theintroduction of a gene capable of being expressed in hematopoietic stemcells which can be transplanted to up to a tertiary host. Use ofconventional NOD-scid mice enabled up to secondary transplant (GuenecheaG. et al., Nature Immunol, 2, 75-82, 2001), and beyond this it was shownthat the present mice were more sensitive and useful as an assay systemfor hematopoietic stem cells.

Example 4 Production of Complete Human Antibodies Using NOG Mice

After NOG mice (8 week-old) and NOD/Shi-scid mice (8 week-old) wereirradiated with 2.5 Gy and 3.5 Gy, respectively, they were subjected tointravenous transplant of 1×10⁶ of CD34⁺ cells which were purified withmagnet beads from human umbilical cord blood (provided by TokaiUniversity, Cell Transplant Research Center, with the consent andapproval of the pregnant women). Specifically, MNC was isolated fromumbilical cord blood using Ficoll, and then CD34⁺ cells were isolated byMACS immunomagnetic separation system (Miltenyl Biotec, Glodach,Germany). After confirming that the CD34⁺ cells had 97% or more purityby FACS, they were used for transplant.

Blood, having been chronologically taken from orbits, and after MNCfraction was obtained by Ficoll, was stained with fluorescence-labeledCD45, CD19, and CD3 antibodies (Becton Dickinson, San Jose, Calif.). Thereconstruction of human lymphocytes, and the ratios of B-cells andT-cells were confirmed with CD45, and fluorescence-labeled CD19 and CD3,respectively, by FACS.

Moreover, thymuses and spleens were extracted from these mice, cellswere prepared therefrom, the cell numbers were counted, and they werestained with various fluorescence antibodies (Becton Dickinson, SanJose, Calif.). Then, they were analyzed by FACS in the same manner asthe above reconstruction confirmation.

From 6 or 8 weeks after the transplant, antigens had been administeredto these mice. As an antigen, DNP-KLH was used. The immunization wasperformed intraperitoneally with 100 μg/mouse of DNP-KLH together withan adjuvant of aluminum hydroxide (ALUM). The same immunization wasperformed every two weeks and serum was taken for measuring antibodytiter by ELISA.

At this juncture, ELISA was performed as follows.

96-well plate was coated with anti human IGs (ICN, Aurora, Ohio) orDNP-KLH, and blocking and washing were performed with 3% BSA. Dilutedanti serum was added thereto. After the reaction at room temperature for2 hours, washing was performed. Then, biotinylated anti-human IgM oranti-human IgG monoclonal antibodies (Phermingen, San Diego, Calif.)were added. After the reaction at 37° C. for 2 hours, washing wasperformed. Then, avidinylated peroxidase was added for 1 hour reactionat room temperature. The plate was washed and TMB peroxidase EIAsubstrate kit solution (Bio-Rad Laboratories, USA) was added for30-minute reaction at room temperature. Then, the reaction was inhibitedwith 10% HCl, and the absorbance was measured at 450 nm. The Igconcentration was calculated in accordance with the standard curve.

(1) Efficiency of Transplanting and Reconstituting CD34⁺ Cells Derivedfrom Umbilical Cord Blood

With respect to the ratio of human CD45⁺ cells in the NOG mice aftertransplanting the umbilical cord blood cells, the ratio of human CD45 inperipheral blood was gradually decreased from 4 weeks onward. However,from 12 weeks onward, suddenly the ratio of increase of T-cells becamehigh, and along with this, it was observed that the ratio of human CD45was also increased in some mice (Table 3). Increases of the CD45⁺ ratio,caused by such T-cell increase, had never been observed in peripheralblood of the existing NOD/Shi-scid mouse. These NOG mice had never beenaffected with GVHD until 14th week.

TABLE 3 Reconstitution of human cells in NOG mice: peripheral blood SCT4 weeks 6 weeks 8 weeks Gp. Source CD34⁺ cells % CD45 % CD19 % CD3 %CD45 % CD19 % CD3 % CD45 % CD19 % CD3 2^(nd) CB2 freeze 1.0 × 10⁶ 47.345.7 1.3 N.A. N.A. N.A. 27.5 42.7 7.2 SCT thawing PBSC1 freeze 1.8 × 10⁶79.5 0.7 13.4 72.5 22.9 47.7 13.3 14.3 65.7 thawing PBSC2 freeze 1.8 ×10⁶ 30.8 3.0 0.1 62.0 63.2 3.8 68.5 93.6 2.1 thawing PBSC3 freeze 1.8 ×10⁶ 26.9 3.7 0.0 N.A. N.A. N.A. 43.5 62.0 7.6 thawing 3^(rd) CB fresh1.8 × 10⁵ 4.8 5.7 0.1 82.2 95.9 0.0 12.4 79.1 1.0 SCT BM freeze 1.9 ×10⁵ 1.7 0.0 0.0 8.2 48.2 0.0 13.5 49.8 0.0 thawing PBSC2 freeze 1.65 ×10⁶  1.4 2.0 0.0 51.6 91.7 0.0 25.6 91.4 0.0 thawing PBSC3 freeze 1.65 ×10⁶  2.1 1.3 0.2 24.1 88.4 0.0 45.3 90.2 0.0 thawing 4th PBSC1 freeze5.0 × 10⁵ 19.4 2.6 0.0 27.7 39.8 0.0 39.2 92.4 0.1 SCT thawing PBSC2freeze 5.0 × 10⁵ 14.5 1.8 0.1 20.0 17.4 0.2 33.4 82.1 0.0 thawing PBSC3freeze 5.0 × 10⁵ 22.1 3.3 0.0 17.5 49.1 0.1 26.1 83.4 0.1 thawing 5^(th)BM freeze 1.5 × 10⁵ 0.9 * * 0.2 * * SCT thawing 6^(th) BMI fresh 3.6 ×10⁵ SCT BM2 fresh 3.6 × 10⁵ PBSC1 freeze 9.0 × 10⁵ thawing PBSC2 freeze9.0 × 10⁵ thawings PBSC3 freeze 9.0 × 10⁵ thawing SCT 10 weeks 12 weeksGp. Source CD34⁺ cells % CD45 % CD19 % CD3 % CD45 % CD19 % CD3 remarks2^(nd) CB2 freeze 1.0 × 10⁶ 19.8 27.9 4.9 SCT thawing PBSC1 freeze 1.8 ×10⁶ Analyzed thawing because of death at 8- week PBSC2 freeze 1.8 × 10⁶13.7 64.5 23.9 10.3 48.4 32.6 thawing PBSC3 freeze 1.8 × 10⁶ 28.4 34.111.1 22.0 35.5 14.0 thawing 3^(rd) CB fresh 1.8 × 10⁵ 21.8 71.7 10.1 SCTBM freeze 1.9 × 10⁵ 7.0 73.7 0.0 mixed thawing PBSC2 freeze 1.65 × 10⁶ 25.8 85.6 0.1 thawing PBSC3 freeze 1.65 × 10⁶  14.3 82.5 0.0 thawing 4thPBSC1 freeze 5.0 × 10⁵ SCT thawing PBSC2 freeze 5.0 × 10⁵ thawing PBSC3freeze 5.0 × 10⁵ thawing 5^(th) BM freeze 1.5 × 10⁵ mixed SCT thawing6^(th) BMI fresh 3.6 × 10⁵ SCT BM2 fresh 3.6 × 10⁵ PBSC1 freeze 9.0 ×10⁵ CD3 thawing eliminated PBSC2 freeze 9.0 × 10⁵ CD3 thawingseliminated PBSC3 freeze 9.0 × 10⁵ CD3 thawing eliminated

(2) T-Cell Differentiation in NOG Mice A. T-Cell Differentiation in theThymus

FIGS. 9A, 9B, 10A and 10B show T-cell differentiation patterns inthymuses. As shown in the figures, CD3 positive cells weredifferentiated in the thymuses of these mice, and it was indicated thatthese cells contained CD4/CD8 DN (Double negative), DP (Doublepositive), and SP (Single positive) as well as those cells in a normalthymus or detected by hu/m-RTOC, and a part of them were mature to beCD1a-low T-cells in 11th or 13th week after transplanting. The number ofthe thymic cells was 1 to 2×10⁶ and this was about one-hundredth of thenumber of thymic cells in a normal mouse. This phenomenon was observedalso in NOD/Shi-scid mice, but its frequency was remarkably low, such as8/28 (28.6%). As opposed to this, NOG mice had a frequency of 7/7(100%).

B. T-Cells at Peripheries

FIGS. 11A, 11B, 12A, 12B, 13A, and 13B show further analysis on T-cellsin splenic cells or peripheral blood. CD3 positive cells were observedeven at the peripheries of the mice from which T-cell differentiationwas observed in their thymuses. These are a group of CD4/CD8 positivecells, and this indicated that there was the possibility that theyincluded cells differentiated in thymuses.

(3) B-Cell Differentiation in NOG Mice A. Subset Differentiation ofB-Cells

Also, FIGS. 14A and 14B show the expression ratios of CD5 by B-cellsamong bone marrow cells. In bone marrow, the differentiation of CD5positive cell, so-called B1a cells, was not facilitated, and groups ofCD19 positive or IgM positive cells were dominant. Further, cells in agroup in which IgM is highly positive expressed CD20. No abnormality wasdetected on the differentiation of human B-cells in the bone marrow ofthese mice. This phenomenon is also observed in B-cell differentiationin NOD/Shi-scid mice, and thus it is considered a common quality.

B. B-Cell at Peripheries

FIGS. 13A and 13B show FACS patterns of B-cells at their peripheries.The ratios of B-cells were almost the same as those in NOD/Shi-scidmice. However, in spleens, CD5 positive cells, so-called B1a cells whichwas a subgroup of B-cells were dominant, and a large difference wasdetected from differentiation patterns in bone marrow. On the otherhand, IgM positive CD5 negative cell groups were also detected at aratio of about 20% and thus this proved that cells, other than B1acells, were also detected.

(4) Antibody Production Ability in NOG Mice

DNP-KLH was administered as an antigen to these mice, and IgM and IgGantibody production amounts were chronologically measured. As a result,antigen non-specific IgM and IgG antigen specific IgM were detected byrepeating the administration three times. Although T-cells were detectedin thymuses and peripheries, antigen specific IgG production was notdetected. (FIGS. 15A, 15B and 15C) FIGS. 15A, 15B and 15C also show theresults of mice having transplanted thereto CD34⁺ cells derived frombone marrow (BM) (FIG. 15B) and peripheral blood (PBSC) (FIG. 15C). Inbone marrow and peripheral blood, there was observed a tendency of lowerproductions of specific IgM and non-specific IgG as compared with theproduction in umbilical cord blood.

The above results indicate that the present mouse differentiates humanT-cells and B-cells and is valuable for use in human antibody productionsystems.

Example 5 Neoplasm Proliferation System

NOD/Shi-scid mice, NOG mice, BALB/cAJc1-nu mice (purchased from CLEA),and C.B-17/Icr-scid mice (purchased from CLEA) were used. All the usedmice were 5 weeks old or older.

As cells to be transplanted to the mice, transplant human tumor cellline, LM-2-JCK was used. LM-2-JCK was a cell line which was establishedfrom lymphoblast lymphoma of a 13-year-old female patient and maintainedby successive heterografts to nude mice. It has been reported thatthough LM-2JCK expresses T-cell antigen CD4 and CD5, it does not expressother cell antigens including antibodies.

A solid tumor which was subcutaneously passaged 12 times in nude micewas used for heterograft assay. The tumor was chopped into pieces inF-10 nutrition supplementary medium (GIBCO BRL) by scissors and fullydispersed by a pipette, and then cell suspension was prepared by passingthrough a nylon mesh. The concentration of viable cells in thesuspension was calculated by trypan blue stain (GIBCO BRL). Aftercentrifugation, tumor cells were dispersed at concentrations of 1×10⁷and 1×10⁶ viable cells/ml into physiological saline. The tumor celldispersion liquid was subcutaneously injected, in an amount of 0.1 mL,at both flanks of mice by using 1 ml syringe equipped with 25 gageneedle. After the injection of the tumor cells, the size of the tumorand the body weight were measured at intervals of one week. On the 21stday after the 1×10⁶ cells were transplanted, when the tumor becamelarge, the mice were sacrificed and the weights and sizes of the tumorswere measured.

The difference in transplantability of LM-2-JCK among the mice havingdifferent backgrounds is shown in Table 4.

TABLE 4 Heterotransplantation of LM-2JCK to mice having differentgenetic backgrounds No. of tumor-engrafted No. of cells Observationmice/No. of Mouse strain grafted period transplanted mice Weight oftumor a) NOG 10⁶ 21 days 10/10 (100%) 3.97 ± 2.10 b) NOD/Shi-scid 10⁶ 21days 10/10/(100%) 1.34 ± 00.77 C.B-17/Icr-scid 10⁶ 21 days 8/10 (80%)1.21 ± 01.06 NOG 10⁵ <9 weeks 8/8 (100%)c) n.t. NOD/Shi-scid 10⁵ <9weeks 5/10 (50%) n.t. C.B-17/Icr-scid 10⁵ <9 weeks 0/10 (0%) n.t. a) Theweight of tumor was measured 21 days after the transplanting andindicated with average ± SD(g). b) As to NOD/Shi-scid andC.B-17/Icr-scid, when p <0.05 (t-test), the significant difference isobserved. c) As to NOD/Shi-scid (p <0.05) and C.B-17/Icr-scid (p <0.01),the significant difference is observed. (x² test) n.t.: not tested

In the case of the transplant of 1×10⁶ cells, all the tumors wereengrafted in NOG mice and NOD/Shi-scid mice. In contrast, theengraftment ratio for C.B-17/Icr-scid was 80%.

The tumor engraftment ratios in these strains were lower in the case ofthe transplant of 1×10⁵ cells, and particularly tumor proliferation wasnot observed in any of the C.B-17/Icr-scid mice.

In the case of the transplant of 1×10⁶ cells, the growth curves of thetumors are shown in FIG. 16.

The tumor proliferation in NOG mice exceeded that of the other 2strains.

On the 21st day, the NOG mice had 2.89 and 3.97 times larger averagetumor volume than the NOD/Shi-scid mice and C.B-17/Icr-scid mice,respectively, had on the same day, and significant differences wereobserved by Student t-test. (p<0.001)

No significant difference was observed in the average volume on the 21stday between tumors engrafted in NOD/Shi-scid mice and tumors engraftedin C.B-17/Icr-scid mice.

Example 6 Establishment of HIV-Infection Model System Using NOG Mice

Establishment of HIV-infection model system using NOG mice was examinedby use of various HIV lines. Typical examples are shown below.

NOG mice were used in this example. For comparison, NOD/Shi-scid micewere also used, which were produced before by backcrossing both mutantsof C.B-17-scid mice and NOD/Shi mice with each other.

HIV-1 used herein was a JRFL virus which was isolated from frontal lobetissue of a patient with AIDS-associated encephalopathy and hadinfectivity on a DNA-cloned macrophage and T-cells. Further, a GFP-HIV-1in which a JRFL virus and a GFP gene were incorporated at env V3 regionand downstream of gp41, respectively was used.

(1) Production of Mice (hu-PBL-NOG Mice) to Which Human MononuclearLymphocytes are Transplanted

1×10⁷ per mouse of peripheral blood human lymphocytes (PBL), provided bya normal person with consent and approval for test use, wereintraperitoneally inoculated directly into NOG mice. For comparison, thePBL from the same donor was intraperitoneally inoculated intoNOD/Shi-scid mice having a normal IL-2Rγ chain.

(2) Infection of Mice with HIV-1

On the 6th day after the PBL inoculation, 1,000 TCID₅₀ of viruses wereitraperitoneally inoculated into mice. The mice were sacrificed 2 weeksafter the virus infection and ascites including abdominal exudates cellswere collected. The number of human cells in the ascites was calculatedby FACS. Further, spleens were extracted and fixed by paraformaldehyde,and then made into paraffin-embedded sections.

(3) Pathological Analysis

The spleen tissue sections were subjected to HE-stain, and further toimmunostaining using antibodies against human CD3, CD4, CD8, andHIV-1p24 antigens. For immunohistologic stain, in addition toconventionally reported ABC method, EPOS method and Envisiont methodwere employed.

The engraftment of human cells in hu-PBL-NOG mice was evaluated.

A series of examination results are shown in Table 5.

TABLE 5 HIV-1 infection test on human PBMC grafted-NOG mice femalewithout TM β1 non existent PBL NOG mice PBL transplantation total PBL160 mL 4.37 × 10⁸ cells 2 × 10⁷ cells per mouse Donor A 14 NOG mice (6males, 8 females) Infection NOG mice JRFL M-tropic 3 males NL4-3T-tropic 3 males JRCSF M-tropic 3 females NLCSFV3EGFP M-tropic 2 femalesMOCK 3 females Cell number × 10⁴ cells % HLA % CD4 % CD8 p24 13^(th) dayafter infection 1. Mock ♀ Ascites 75 67.7 21.4 56.4 Spleen 510 71.8 13.845.5 PBL 430 77.9 16.8 49.9 0 2. Mock ♀ Ascites 130 67.6 14.8 60.5Spleen 698 63.3 12.7 49.2 PBL 86 66.6 11.6 55.5 0 3. Mock ♀ Ascites 11062.3 36.4 44.2 Spleen 434 61.3 20.9 45.2 PBL 93 34.7 20.6 44.7 0 HIV-1infection to human PBMC grafted-NOG mice (2) 4. JRFL ♂ Ascites 152 75.51.7 53.9 Spleen 335 54.6 2.8 64.6 PBL 210 42.8 2.1 77.3 3,090 5. JRFL ♂Ascites 220 75.8 1.8 57 Spleen 12 75.8 1.65 79.6 PBL 870 81.1 1.8 77.95,445 6. JRFL ♂ Ascites 178 36.3 1.79 54.1 Spleen 418 65.3 1.1 74.4 PBL65 33.1 1.72 75.6 1,961 7. NL4-3 ♂ small spleen Ascites 120 7.6 9.7474.1 Spleen 14 no FACS PBL non-existent 8. NL4-3 ♂ Ascites 84 75.5 1.4345.7 Spleen 465 53 1.64 60.0 PBL 26 11.3 4.59 49.3 118 9. NL4-3 ♂Ascites 110 8.1 9.33 80.6 Spleen 17 14.8 19.1 23.5 PBL 3 0.6 13.0 27.8 0for three male NL4-3, their ears were excised. 10. JRCSF ♀ Ascites 32029.9 0.77 93.7 Spleen 230 67 1.85 74.9 PBL 49 33.7 0.48 71.7 3,132 11.JRCSF ♀ Ascites 63 68.5 3.18 41.6 Spleen 390 68.7 3.58 85.8 PBL 121 2.94.5 31.5 4,180 12. JRCSF ♀ poor construction Ascites 110 15.2 23.0 62.4Spleen 380 14.5 2.98 89.8 PBL 110 44.7 2.06 89.4 864 13. NLCSFV3EFFP ♀GFP+ of spleen and PBL are 0.1% or less Ascites 43 72.5 2.26 69.8 Spleen250 50.8 0.77 67.7 PBL 124 76.5 5.07 63.3 271 14. NLCSFV3EGFP ♀ GFP+ ofascites, spleen and PBL are 0.2 to 0.3% or less Ascites 110 44.3 6.3676.3 Spleen 560 2.7 23.3 44.4 PBL 127 3.4 5.26 84.2 114

CD4 positive cells were specifically killed by all the HIV viruses.

Conventionally, it has been reported that only M-tropic viruses canproliferate in mice and CD4 positive cells are killed, but T-tropicviruses can proliferate in NOG mice.

It was found that human CD4 positive T-cells and CD8 positive T-cellswere each engrafted around central arteries of spleens of the mice towhich human peripheral blood was transplanted. With respect to thenumber of human CD3 positive cells, NOG mice had obviously a largernumber of human cells than NOD/Shi-scid mice. According to themeasurement by FACS, there were 1×10⁷ or more human CD4 positive cellson average in ascites.

On the other hand, CD4 positive cells were hardly detected fromNOD/Shi-scid mice and almost all the detected cells were CD8 positivecells. In the example previously described, the antibodies (TMβ-1) (0.5mg per mouse) which were against mouse IL-2 receptor β chain andsuppressed the differentiation of NK cells were intraperitoneallyinjected into the NOD/Shi-scid mice 3 days before the inoculation ofhuman PBL. However, this treatment was not conducted in this example. Ithas been already confirmed in other tests that antibody-treated micewould have many CD4 and CD8 positive human cells engrafted to them.Still though the number of human cells from NOG mice was apparently 3 to4 times more than the number of cells from NOD/Shi-scid mice that hadbeen subjected to the antibody treatment.

In the HIV-infected mice, p24 positive cells which were HIV antigenswere detected around spleen arteries. Further, in spleens and ascites ofthe HIV-infected mice which express GFP, cells with GFP expression wereclearly detected. The number of virus-infected cells of NOG mice wasapparently larger than that of NOD/Shi-scid mice.

Example 7 Establishment of HTLV-1 Infection Model System Using NOG Mice

1×10⁷ cells per mouse of MT-2 cells which were cell lines derived fromHTLV-1 leukemia, were transplanted to NOG mice.

In the 4th week after the transplant, the mice having lymphomas formedtherein (Table 6) were counted, and it was checked whether or not themice having lymphomas were infected with a HTLV gene, by Southernblotting and PCR. As a result of a pathological search, dermal lymphomaand posterior intraperitoneally bilateral lymphomas are observed. Themice having a large tumor had metastasis in lymph nodes around thestomach and had cancer cells infiltrated in a pleura thereof. Further,HTLV positive cells increased in peripheral blood. This indicated atypical onset of leukemia. Moreover, lung-interstitial invasion was alsoobserved. All of these cells expressed Tax protein, but there was nointracerebral invasion.

TABLE 6 No. of mice having Cell treatment by lymphoma (after 1 No. ofmice TMβ-1 gamma ray month) NOD/Shi-scid mice 5 — — 0 NOG mice 40 — —33(82.5%) NOG mice 4 — 20,000 rad 0 NOG mice (no cell 2 — — 0transplanted) 1. Transplantation of HTLV-infected cells to NOG mice 2 ×10⁷ cells per mouse

Example 8 Examination of Leukemic Changes by Introducing NeoplasticCells and Transplantability of HTLV-1 Positive Cells Using NOG Mice

Transplant tests of various HTLV-1 cell lines and tumor formation testswere performed on NOG mice and the usefulness of the mice was examined.

Using 3 samples of HTLV-1 leukemia derived cell lines [L=Leukemic celllines: ED-40515(−), MT-1 and TL-Oml], 6 samples of HTLV-1 infected celllines [IT=infected transformed cell lines: SLB-1, M8116, HUT-102, MT-2,MT-4 and TY9-31MT], 10⁷⁻⁸ cells/0.5 mL were transplanted to NOG mice andNOD/Shi-scid mice subcutaneously at a left posterior auricle thereof,and to some of them subcutaneously at a gluteal region thereof. Then,the tumor size in the mice, tissue image, and the mice with, and thosewithout, leukemia were chronologically compared for examination, and therelations between these and transcription factors such as NFkB orfluctuation of Tax protein were also analyzed.

Within 2 weeks, a tumor with a size of 24×17×11 mm was formed in allcases of HTLV-1 leukemia derived cell lines [L] and exceptionally inSLB-1 of HTLV-1 infected cell line [IT]. In contrast, in 5 out of 6cases of IT, a tumor with a size of at most 10×10×7 mm was formed, andin the case of M8116 or TY9-31MT, there was almost no tumor formation.(Table 7)

TABLE 7 Development of HTLV-1 infected-cells and leukemia cells in NOGmice and their features in vitro and in vivo In vitro In vivo No. ofproliferation II-2 Tax Cell Route and site of period size of tumor Cellline Cell type mice pattern dependence expression inoculated (×10⁷)inoculation (days) (mm) ED-40516(−) l 1 separately − − 7.5 I.m Buttock16 23 × 18 × 12 7 separately − − 7.5 s.c post-auricular 15-16 25 × 18 ×12 3 separately − − 4 s.c post-auricular 16 25 × 18 × 12 3 separately −− 1 s.c post -auricular 15 22 × 17 × 10 SLB-1 IT 1 clustered − + 7.5 I.mButtock 15 20 × 10 × 18 7 clustered − + 7.5 s.c post-auricular 15-16 20× 10 × 18 3 clustered − + 5 s.c post-auricular 15 21 × 12 × 17 MT-1 L 1separately − − 7.5 I.m Buttock 20 25 × 15 × 10 1 separately − − 7.5 s.cpost-auricular 20 25 × 15 × 10 3 separately − − 4 s.c post-auricular 2423 × 16 × 12 TI-oml L 1 separately − − 7.5 I.m Buttock 20 25 × 15 × 10 1separately − − 7.5 s.c post-auricular 20 25 × 15 × 10 3 separately − − 4s.c post-auricular 20 22 × 23 × 8  M8116 IT 1 clustered − + 7.5 I.mButtock 18 sesame 2 clustered − + 7.5 s.c post-auricular 18 sesaneHUT-102 IT 1 clustered − + 7.5 I.m Buttock 18 10 × 10 × 7  2 clustered− + 7.5 s.c post-auricular 18 10 × 10 × 7  MT-2 IT 1 clustered − + 7.5I.m Buttock 18 10 × 10 × 7  2 clustered − + 7.5 s.c post-auricular 18 10× 10 × 7  MT-4 IT 1 clustered − + 7.5 I.m Buttock 18 5 × 5 × 3 2clustered − + 7.5 s.c post-auricular 18 5 × 5 × 3 TY9-31MT-2 IT 1clustered − + 7.5 I.m Buttock 18 sesame 2 clustered − + 7.5 s.cpost-auricular 18 sesame L: leukemia cell line IT: infected-tranformantcell line

The case of this tumorigenic cell line showed that the degree of theemergence of leukemic cells in peripheral blood and the invasion oforgans were relatively high, but some cases of nontumorigenic cell lineshad higher degrees thereof (e.g. HUT-102, MT-2 etc.). Thus, there werenot necessarily correlations between tumorigenicity and the degrees ofthe emergence of leukemic cells in peripheral blood and the invasion onorgans. (Table 8).

TABLE 8 Infiltration of leukemia cells into various organs of NOG miceperipheral bone Cell strain Tumor blood (PB) marrow (BM) liver spleenlung brain kidney heart Ed-40515(−) +++ ++ − + + +− Spongy ++ − +− SLB-1+++ +++ + ++ + Granular Spongy + + +++ ++ MT-1 +++ ++ − + + + Spongy − −+− TI-oml +++ ++ − Giant + +− − − − +++ M8116 + + − − − − − − − Hut-102++ ++ +++ Nodule +++ Pneumonia Spongy − − +++ + +− MT-2 ++ ++ +− +− +++− − + − MT-4 ++ + +− − ++ − − − − TY9-31MT-2 +− − − − − − − − −

10⁷ cells of tumorigenic ED-40515(−) were transplanted subcutaneously ata left posterior auricle of NOG mice and NOD/Shi-scid mice and they werecompared in tumorigenicity in the second week. A tumor with a size of22×17×10 mm was formed in the NOG mice, and in contrast a tumor washardly formed at all in NOD/Shi-scid mice. This revealed that NOG micewere prone to tumor formation. (Table 9)

TABLE 9 Comparison of tumor formation between NOG mice and NOD/Shi-scidmice to which ED- 40515(−) cell type was transplanted Survival periodMouse strain No. of mice Cells inoculated (×10⁷) (days) Tumor NOG mouse3 1 15 +++ NOD/Shi-scid 3 1 15 − mouse +++ tumor large enough tovisually observe − tumor which cannot visually be observed

For the purpose of investigating whether or not the NOG mice are proneto tumor formation in B-cell type, as well as in T-cell type, 7×10⁷ ofBJAB cells of which only EBER (EBV-Encoded Small RNA) and a vector weretransformed, were transplanted subcutaneously at a left posteriorauricle of NOG mice. In the 3rd week, they were compared intumorigenicity. A significantly large tumor (26×18×7/13×18×3 mm) wasformed in the mice with BJAB-EBER compared to the mice with BJAB-VECTOR.The tumor size thereof was comparable to that in the case of ED-40515(−)in the above example. (Table 10)

TABLE 10 Proliferation of BJAB-EBER cell line and BJAB-VECTOR cell linein NOG mice Gene No. of Cells Route and site of Survival Tumor Weight ofCell line introduction Mice inoculated (×10⁷) inoculation period (days)size (mm) tumor BJAB-EBER gene 3 7 s.c. post-auricilar 21 26 × 18 × 72.86 g introduced BJAB-VECTOR gene not 3 7 s.c. post-auricular 21 13 ×18 × 3 0.73 g introduced

Assuming that effective tumor formation by 10⁷ of ED-40515(−) in NOGmice was attributable to active neogenesis of tumoral vessels via CXCR4of tumor cells, it has been expected that tumor formation would beinhibited as long as there was continuous administration of KRH-1636, acompetitive agent of SDF-1 which was a ligand thereof in endothelialcells. However, in the case of ED-40515(−) and SLB-1 (histologicallyangiotropic) accompanied by bleeding, the mice formed tumors with sizesof 25×18×12 mm and 20×10×18 mm equivalent to those of non-treated group,even though KRH-1636 was intraperitoneally administered every day.

TABLE 11 Effect of CXDR4 antagonists on In Vivo Growth and Proliferationof HTLV-1 infected cell lines in NOG mice No. of Cells Route of drugSurvival Tumor Cell line group mice inoculated (×10⁷) administrationperiod (days) size (mm) Remarks ED-40515(−) Drug 3 7.5 intraperitoneally15 25 × 18 × 12 progressive large tumor KRH-1636 0.14 mg/mouse Control 37.5 intraperitoneally 15 25 × 18 × 12 progressive large tumor medium 0.2ml/mouse SLB-1 Drug 3 7.5 intraperitoneally 15 20 × 10 × 18 progressivelarge tumor KRH-1636 with hemorrhage 0.14 mg/mouse Control 3 7.5intraperitoneally 15 20 × 10 × 18 progressive large tumor medium withhemorrhage 0.2 ml/mouse

Using Cytospin specimens of in vitro culture cells and frozen sectionspecimens of in vivo tumor formative cells, both from ED-40515(−) andSLB-1, the immunostaining manners of CD4, CD8, CD3, CXCR4, CCR5 andSDF-1 were comparatively tested by enzyme antibody technique. All thecases of CD4, CD3, CXCR4 and SDF-1 were almost equally positive, and allthe cases of CCR5 were negative. CD8 was negative in vitro, but positivein vivo. (Table 12)

TABLE 12 In vitro and in vivo examination of HTLV-1 infected cell line-transplanted NOG mice by FACS, WB, EMSA and immunohistochemistryImmunohistochemistry CD4 CD8 CD3 CXCR4 CCR5 SDF-1 In-vitro ED-40515(−)++ − + + − ++ SLB-1 ++ − + + − ++ In-vivo ED-40515(−) +++ + + + − +++SLB-1 +++ + + + − +++ +++ strongly positive ++ positive + weaklypositive − negative

Using in vitro culture cells and in vivo tumor formative cells ofED-40515(−), Table 13 Western blotting analyses on Tax, CXCR4, OX40 andOX40L were performed. All the cases of Tax were negative. However, allthe cases of CXCR4, OX40 and OX40L were positive, and there was nosignificant difference in their strengths. (Table 13)

In vitro and in vivo examination of HTLV-1 infected cellline-transplanted NOG mice by FACS, WB, EMSA and immunohistochemistry

In-vitro In-vivo WB TAX CXCR4 OX40 OX40L TAX CXCR4 OX40 OX40LED-40515(−) − ++ + + − + + + SLB-1 ++ ++ +++ −

Using in vitro culture cells and in vivo tumor formative cells ofED-40515(−), transcription factor activity of NFkB was examined byelectrophoretic mobility shift assay (EMSA), though no differencetherebetween was observed. (Table 14)

TABLE 14 In vitro and in vivo examination of HTLV-1 infected cellline-transplanted NOG mice by FACS, WB, EMSA and immunohistochemistryIn-vitro In-vivo EMSA NFkB NFkB ED-40515(−) +++ +++ SLB-1 +++

The following points were recognized in the system wherein thetransplant was conducted subcutaneously at posterior auricles of the NOGmice.

(1) It was certainly confirmed that large tumors were formed in the NOGmice within very short periods, like 15 days after innoculation, whichhad not been expected. Conversely, NOD/Shi-scid mice (IL-2R γchain^(+/+)) did not form any tumors.

(2) It was revealed that the NOG mice genetically delete NK cells, andtherefore they did not require the pre-treatment against anti NK cellsusing monoclonal antibodies etc., which is essential in the case ofC.B-17/Icr-scid or NOD/Shi-scid mice. (IL-2R γ chain^(+/+))

(3) The transplant was performed subcutaneously at a posterior auricle,that is, selecting a site which has a anatomically lower number of NKcells than an intraperitoneal site, and this allows a tumor to be easilyformed. As a result, the size of the tumor was easily observed byappearance without need for incision.

(4) Although Uchiyama et al. reported that MT-1, T-2 and TL-Oml did notform any tumors, in the present case relatively small tumors were formedin them. (Imada K, Takaori-Kondo A, Akagi T, Shimotohno K, Sugamura K,Hattori T, Yamabe H, Okuma M, Uchiyama T: Tumorigenicity of human T-cellleukemia virus type I-infected cell lines in severe combinedimmunodeficient mice and characterization of the cells proliferating invivo. Blood 86:2350-7, 1995, Uchiyama, T: Human T cell leukemia virustype I (HTLV-I) and human diseases Annu Rev Immunol 15:15-37, 1997).

Therefore, the system wherein the transplant is carried outsubcutaneously at posterior auricles of the NOG mice, is an innovativetumor transplant system. Further, it was revealed that it could work inthe same manner on B-cell tumors as well as on T-cell type tumors.Namely, these indicate also that this system is a valuable transplantsystem for the transplant of cancer cells or human normal lymphoidtissues.

In addition, when there is no laboratory animal model or it isdifficult, even if there is, to put one into practical use, a humandisease model can be established by transplanting human cells or tissuesto this NOG mouse, thereby enabling disease researches, for which therehas been no other choice but to be dependent on in vitro tests, with atest system which comes close to a limitless human in vivo test. Thus,this mouse is considered to make great contributions to the elucidationof mechanisms of disease onset, development of therapies etc. Forexample, irrespective of potent HAART therapy against HIV-1 infection,the rebound or mutant virus emergence of viremia is of primaryimportance. The reservoir of infectious viruses thereof is known to beFDC (follicular dendritic cells) of lymphatic follicle, and forresearches on it, it is indispensable to establish a humanized modelmouse to which lymphoid tissues including human lymphatic follicle aretransplanted. For this, the present NOG mouse is useful.

Furthermore, with respect to a cause to make this system prone to form atumor, examinations were made on what factor is increased or activatedin transplanted cells, as compared with in vitro culture cells. All thecell lines used in the above examples were IL-2 independent, IL-2producing, further Tax and CXCR4-SDF-1 system were revealed not to bedirectly associated therewith. However, within the search the presentinventors have made, there was no factor except the above mentionedwhich the inventors recognized indicated a significant difference incomparison between in vitro culture cells and in vivo tumor-formingcells.

INDUSTRIAL APPLICABILITY

The present invention provides a method of producing NOG mice moresuitable for engraftment of heterologous cells, particularly humancells, and a mouse produced by the method, as compared with anNOD/Shi-scid mouse and an NOD/LtSz-scid, β2m null mouse bothconventionally known as an immunodeficient mouse. Transplanting humanstem cells to the thus obtained mice enables a human stem cell assaysystem to be established. Further, transplanting human cells responsiblefor immunity enables human antibodies to be produced using the mouse ofthe present invention. Furthermore, a human tumor model mouse can beproduced by transplanting and engrafting human tumors to the mouse, andthe mouse can be used for therapies for tumors, screening of therapeuticagents and the like. Moreover, it is possible to produce an HIV orHTLV-1 infected model mouse to which an HIV or HTLV1 infected humanlymphocyte is engrafted, and researches can be made on in vivoproliferation mechanism of HIV-1, HTLV-1 etc. Also, it is possible toconduct development of a therapy for virus infection, screening of atherapeutic agent for virus infection or the like.

All publications cited herein are incorporated herein with the wholecontents thereof. Further, it is easily understood by those skilled inthe art that various changes and modifications can be made in theinvention without departing from the technical idea and scope describedin the appended claims. It is intended that the present invention coverall such changes and modifications.

The invention claimed is:
 1. A method of producing a human antibodycomprising: introducing human B-cells, or both human B cells andT-cells, or human stem cells which differentiate into human B-cells orboth human B cells and T-cells, into a mouse produced by a methodcomprising backcrossing a mouse B with a mouse A, wherein said mouse Ais a NOD-scid/scid mouse, and wherein said mouse B is an interleukin2-receptor γ chain gene knockout mouse, wherein said mouse produced bythe method retains the human B-cells or both human B cells and T-cells;immunizing said mouse with an antigen; and collecting the human antibodyproduced from the mouse.
 2. A method of producing an antibody-producingcell line which produces a human antibody, comprising: introducing humanB-cells, or both human B cells and T-cells, or human stem cells whichdifferentiate into human B-cells or both human B cells and T-cells, intoa mouse produced by a method comprising backcrossing a mouse B with amouse A, wherein said mouse A is a NOD-scid/scid mouse, and wherein saidmouse B is an interleukin 2-receptor γ chain gene knockout mouse,wherein said mouse produced by the method retains the human B-cells orboth human B cells and T-cells; immunizing said mouse with an antigen;collecting from the mouse a cell which produced the antibody against theantigen; and establishing a cell line of the cell.
 3. The method ofclaim 1, wherein the human B-cells, or both human B cells and T-cells,or human stem cells which differentiate into human B-cells or both humanB cells and T-cells, are introduced intravenously.
 4. The method ofclaim 1, wherein the human stem cells which differentiate into humanB-cells or both human B cells and T-cells are CD34+ cells.
 5. The methodof claim 1, wherein the human stem cells which differentiate into humanB-cells or both human B cells and T-cells are cord blood cells.
 6. Themethod of claim 2, wherein the human stem cells which differentiate intohuman B-cells or both human B cells and T-cells are cord blood cells. 7.The method of claim 2, wherein the antibody-producing cell is collectedfrom the spleen.
 8. The method of claim 2, wherein theantibody-producing cell is collected from a lymph node.